In the commercial production of crops, it is desirable to easily and quickly eliminate unwanted plants (i.e., “weeds”) from a field of crop plants. An ideal treatment would be one which could be applied to an entire field but which would eliminate only the unwanted plants while leaving the crop plants unharmed. One such treatment system involves the use of crop plants that are tolerant to a herbicide. When the herbicide is sprayed on a field of herbicide-tolerant crop plants, the crop plants continue to thrive while non-herbicide-tolerant weeds are killed or severely damaged.
Crop tolerance to specific herbicides can be conferred by engineering genes into crops which encode appropriate herbicide metabolizing enzymes. In some cases these enzymes, and the nucleic acids that encode them, originate in a plant. In other cases, they are derived from other organisms, such as microbes. See, e.g., Padgette et al. (1996) “New weed control opportunities: Development of soybeans with a Round UP Ready™ gene” and Vasil (1996) “Phosphinothricin-resistant crops,” both in Herbicide-Resistant Crops, ed. Duke (CRC Press, Boca Raton, Fla.) pp. 54-84 and pp. 85-91. Indeed, transgenic plants have been engineered to express a variety of herbicide tolerance genes from a variety of organisms, including a gene encoding a chimeric protein of rat cytochrome P4507A1 and yeast NADPH-cytochrome P450 oxidoreductase (Shiota et al. (1994) Plant Physiol. 106: 17), among other plant P450 genes (see, for example, Didierjean, L. et al. (2002) Plant Physiol. 130: 179-189; Morant, M. S. et al. (2003) Opinion in Biotechnology 14:151-162). Other genes that confer tolerance to herbicides include: acetohydroxy acid synthase (“AHAS”), which has been found to confer resistance to multiple types of ALS herbicides on plants expressing it and has been introduced into a variety of plants (see, e.g., Hattori et al. (1995) Mol. Gen. Genet. 246: 419); glutathione reductase and superoxide dismutase (Aono et al. (1995) Plant Cell Physiol. 36: 1687); and genes for various phosphotransferases (Datta et al. (1992) Plant Mol. Biol. 20: 619).
While herbicide-tolerant crop plants are presently commercially available, improvements in every aspect of crop production are continuously in demand. Herbicides and crops that are presently commercially available unfortunately have particular characteristics which can limit their usefulness in commercial crop production. Particularly, individual herbicides have different and incomplete spectra of activity against common weed species.
The acetolactate synthase, or ALS (also known as AHAS) family of herbicides control weeds by inhibiting the production of branch chain of amino acids that are essential to plant growth and development. Specifically, they bind to the plant ALS enzyme. Commonly used herbicides in this family include nicosulfuron, rimsulfuron, and chlorsulfuron, among others. Herbicides in this category can be quite crop-specific. Embodiments of the invention relate to plants that are resistant to members of the ALS-inhibiting class of herbicides, which encompasses 5 sub-classes of herbicides including, but not limited to, the sulfonylurea (SU) family of herbicides and the imidazolinone family of herbicides.
The pigment synthesis-inhibiting class of herbicides targets the enzymes that allow plants to synthesize pigments, such as carotenoid pigments or chlorophyll pigments. Loss of pigment results in photo-destruction of chlorophyll and whitening of plant tissues, which is why these herbicides are often called “bleaching” herbicides. An example of a sub-class of the bleaching herbicides is the HPPD-inhibiting class, which inhibits the 4-hydroxyphenylpyruvate dioxygenase (HPPD) enzyme (Lee et al. (1997) Weed Sci. 45:601-609). Herbicides in this family include, but are not limited to, mesotrione, tembotrione, topramezone and sulcotrione, among others. Corn is generally tolerant to mesotrione due to metabolism of the herbicide (Mitchell et al. (2001) Pest Mgt. Sci. 57:120-128). The same detoxification system may give tolerance to both mesotrione and some SU herbicides (Green & Williams (2004) Proceedings Weed Science Society of America 44:13). Embodiments of the invention relate to plants that are resistant to members of the pigment synthesis-inhibiting class of herbicides.
The protoporphyrinogen oxidase (PPO)-inhibiting class of herbicides interferes with the synthesis of chlorophyll, resulting in compounds that produce highly active compounds (free-radicals). These reactive compounds disrupt cell membranes which results in the leaf burning associated with post-emergence applications of these products. Herbicides in this family include, but are not limited to, acifluorfen, fomesafen, lactofen, sulfentrazone, carfentrazone, flumiclorac and flumioxazin, among others. Embodiments of the invention relate to plants that are resistant to members of the PPO-inhibiting class of herbicides.
Photosystem II (PSII)-inhibiting herbicides have a mode of action that involves interaction with components in the electron transfer chain of Photosystem II. Photosynthesis requires the transfer of electrons from Photosystem II to Photosystem I. A key step in this electron transfer chain is the reduction of plastoquinone (PQ) by the D1 protein in the thylakoid membrane. PSII-inhibitor herbicides bind to the D1 protein, thus inhibiting PQ binding and interrupting the electron transfer process. This results in the plants not being able to fix carbon dioxide and produce the carbohydrates needed for the plant to survive. The block in electron transfer also causes an oxidative stress and the generation of radicals which cause rapid cellular damage. PSII-inhibiting herbicides are represented by several herbicide families, including the symmetrical triazines, triazinones (asymmetrical triazines), substituted ureas, uracils, pyridazinones, phenyl carbamates, nitrites, benzothiadiazoles, phenyl pyridazines, and acid amides. Embodiments of the invention relate to plants that are resistant to members of the PS II-inhibiting class of herbicides.
Synthetic auxin herbicides are a widely used class of herbicides that mimic the natural auxin hormones produced by plants. Auxins regulate many plant processes, including cell growth and differentiation. Auxins are generally present at low concentrations in the plant. Synthetic auxin herbicides mimic natural auxins and cause relatively high concentrations in the cell that result in a rapid growth response. Susceptible plants treated with these herbicides exhibit symptoms that could be called ‘auxin overdose’, and eventually die as a result of increased rates of disorganized and uncontrolled growth. Embodiments of the invention relate to plants that are resistant to members of the synthetic auxin class of herbicides.
Some embodiments of this invention are based on the fine mapping, cloning and characterization of the gene responsible for an important herbicide resistance mechanism in maize.
It has been known since the early 1990s that natural tolerance in maize (Zea mays L.) to a subset of sulfonylurea herbicides (nicosulfuron [Dupont Accent® herbicide], rimsulfuron, primisulfuron, and thifensulfuron) is controlled by a single gene (named nsf by Kang (1993) Journal of Heredity 84(3): 216-217), with resistance dominant and sensitivity recessive (Harms et al. (1990) Theor. Appl. Genet. 80:353-358; Kang (1993) supra; Green & Uhlrich (1993) Weed Sci. 41:508-516; Green & Uhlrich (1994) Pestic. Sci. 40:187-191). It is also known that tolerant maize plants metabolize nicosulfuron by hydroxylation, with the characteristics of a cytochrome P450 (Forme-Pfister et al. (1990) Pesticide Biochem. Physiol. 37:165-173; Brown & Cotterman (1994) Chem. Plant Prot. 10:47-81). It has been suggested that the same corn gene responsible for determining tolerance to some sulfonylurea herbicides is also responsible for the tolerance to bentazon (Barrett et al. (1997) Role of cytochrome P-450 in herbicide metabolism and selectivity and multiple herbicide metabolizing cytochrome P-450 activities in maize. In K. K. Hatzios, ed. Regulation of Enzymatic Systems Detoxifying Xenobiotics in Plants. Dordrecht: Kluwer Academic. pp. 35-50; Green (1998) Weed Technology 12:474-477) and HPPD inhibitor herbicides such as mesotrione (Green & Williams (2004) supra; Williams et al. (2005) HortScience 40(6):1801-1805). Recent advances in the development of the maize physical map and integrated markers (Bortiri et al. (2006) Curr Opin Plant Biol. 9(2):164-71) has allowed a positional cloning approach to be used for identifying the Nsf1 locus.
The Nsf1 resistance gene of the embodiments of the present invention encodes a novel gene related to the cytochrome P450 family. While multiple cytochrome P450 genes have been described, they differ widely in their response to different pathogens and exact action. The novel cytochrome P450 gene described in this disclosure has been demonstrated to provide improved tolerance or resistance to numerous herbicides, including nicosulfuron, rimsulfuron, primisulfuron, thifensulfuron and mesotrione.